1. Field of the Invention
The present invention is generally directed to methods of identifying highly angiogenic endothelial cells. In particular, the present invention is directed to a method of screening endothelial cells for angiogenic capability comprising quantifying the endothelial cells that are VEGF R2 positive and CD34 positive, the VEGRF 2 amount, or determining the VEGF R2 to VEGF R1 ratio or number of VEGF-R2 positive cells to number of VEGF-R1 positive cells.
2. Background of the Invention
Endothelial cells that form the lining of blood vessels are well known for their capacity to adjust their numbers and arrangement to suite local requirements. All tissues depend on a blood supply and the blood supply depends on endothelial cells. Blood vessels create an adaptable life support system in every region of the body. If not for endothelial cells extending and maintaining this network of blood vessels, tissue growth and repair would not be possible.
The largest blood vessels are arteries and veins, which have a thick tough outer wall of connective tissue and smooth muscle. The wall is lined by a thin single layer of endothelial cells, separated from the surrounding outer layers by a basal lamina. While the amounts of connective-tissue and smooth muscle in the vessel wall may vary according to the vessel's diameter and function, the endothelial lining is always present. In the smaller capillaries and sinusoids, the walls consist solely of endothelial cells and basal lamina. Thus, endothelial cells line the entire vascular system. Studies have shown that arteries and veins develop from small vessels constructed solely of endothelial cells and a basal lamina, connective tissue and smooth muscle being added later where required upon signals from the endothelial cells.
Throughout the vascular system endothelial cells retain a capacity for cell division and movement. The is important in repair and maintenance of the vascular system. For example, if a part of the wall of a blood vessel is damaged and loses endothelial cells, neighboring endothelial cells will proliferate and migrate in to cover the exposed surface. Newly formed endothelial cells have also been know to cover the inner surface of plastic tubing used by surgeons to replace damaged blood vessels.
Endothelial cells not only repair damaged blood vessels, they also create new blood vessels. They do this in embryonic tissues to support growth, in normal adult tissue for repair and maintenance, and in damaged tissue to support repair. This process is called angiogenesis.
Angiogenesis is the fundamental process by which new blood vessels are formed. This process is essential in numerous normal physiological phenomena such as embryonic development, tissue growth, tissue remodeling, and wound healing. Angiogenesis is also important in certain pathological events. In addition to a role in solid tumor growth and metastasis, other notable conditions with an angiogenic component are arthritis, psoriasis and diabetic retinopathy. In other pathologies such as the cardiovascular diseases, the diseases of the peripheral arteries as well as the vascular and cerebral lesions, angiogenesis can present an important therapeutic base. The promotion of angiogenesis in the damaged locations can lead to formation of sanguineous neovessels that are lateral and alternative to the damaged vessels, thereby providing blood and, thus, oxygen and other nutritive and biological factors necessary for the survival of the tissues involved.
During the process of angiogenesis, endothelial cells, which exist in a quiescent state as part of an existing blood vessel, grow and enter a migratory, proliferative state. This migratory, proliferative state is eventually resolved when the cells differentiate into capillary tubes and return to the quiescent state as part of a functional new blood vessel. The process of angiogenesis is orchestrated by a complex network of multiple macromolecular interactions. Some essential angiogenic factors include fibroblast growth factor-basic (bFGF), vascular endothelial growth factor (VEGF), the angiopoietins, cytokines, extracellular matrix proteins, and matrix metalloproteases. These factors are produced locally by stromal cells (e.g., smooth muscle cells, pericytes, fibroblasts) and by activated leukocytes that are recruited to the area. A feature of endothelial cells is their capacity to undergo tubulogenesis, an aspect of angiogenesis, under appropriate conditions.
Angiogenesis is regulated in both normal and malignant tissues by the balance of angiogenic stimuli and angiogenic inhibitors that are produced in the target tissue and at distant sites. Vascular endothelial growth factor-A (VEGF, also known as vascular permeability factor, VPF) is a primary stimulant of angiogenesis. VEGF is a multifunctional cytokine that is induced by hypoxia and oncogenic mutations and can be produced by a wide variety of tissues.
Angiogenesis is stimulated and harnessed by some neoplasms (e.g., tumors) to increase nutrient uptake. However, in contrast to normal angiogenesis, which leads to anastomoses (i.e., vessel connections) and capillary maturation, angiogenesis associated with neoplasia is a continuous process. Endothelial cells are activated by nearby neoplastic cells to secrete not only VEGF which stimulates angiogenesis, but also matrix metalloproteases (MMP) which degrade the surrounding extracellular matrix. The endothelial cells then invade the extracellular matrix where they proliferate, migrate, and organize to form new blood vessels, which support neoplasm growth and survival.
The newly vascularized neoplasm continues to grow, leading to further nutrient deprivation and chronic pro-angiogenic signaling. The vasculature of neoplasms is characterized by the presence of structural irregularities (lacunae) and a low rate of formation of inter-vessel connections. This partially dysfunctional vasculature fuels the permanent requirement for angiogenesis. Additionally, this incomplete vasculature is believed to promote the shedding of neoplastic cells into the systemic circulation. Hence, the angiogenic potential of a neoplasm correlates with metastatic potential. As a significant proportion of neoplasms are dependent on continued angiogenesis, inhibition of angiogenesis blocks neoplasm growth which often leads to complete necrosis of the neoplasm.
The interplay of growth factors (e.g., VEGF) and surface protein extracellular interactions drive the process of angiogenesis through a predictable sequence of events. Activation of endothelial cells by pro-angiogenic stimuli results in vasodilation, hyperpermeability, and local release of proteases which degrade the basement membrane and extracellular membrane (ECM). This allows the formation of a provisional fibrin matrix, which provides a primary scaffold for the assembly of early microvessels. Motogenic endothelial cells sprout into the matrix and migrate with controlled matrix degradation at the tip. Proliferation occurs proximal to migration with formation of a primitive tube. Extensive remodeling ensues until the new capillary matures and anastomoses (i.e., fuses and joins) with other sprouts.
VEGF is critical to the angiogenesis process. VEGF induces angiogenesis and endothelial cell proliferation and it plays an important role in regulating vasculogenesis. VEGF is a heparin-binding glycoprotein that is secreted as a homodimer of 45 kDa. Most types of cells, but usually not endothelial cells themselves, secrete VEGF. VEGF is known to increase vascular permeability and cause vasodilatation, partly through stimulation of nitric oxide synthase in endothelial cells. VEGF can also stimulate cell migration and inhibit apoptosis. There are three receptors in the VEGF receptor family (VEGF R1, VEGF R2 and VEGF R3). These receptors have the common properties of multiple immunoglobulin G (IgG)-like extracellular domains and tyrosine kinase activity. Endothelial cells also express additional VEGF receptors, Neuropilin-1 and Neuropilin-2. VEGF-A is the most commonly occurring VEGF. VEGF-B through VEGF-D are lesser occurring. VEGF-A binds to VEGF R1 and VEGF R2 and to Neuropilin-1 and Neuropilin-2. Placental Growth Factor (PIGF) and VEGF-B bind VEGF R1 and Neuropilin-1. VEGF-C and -D bind VEGF R3 and VEGF R2. VEGF R1 and VEGF R2 are upregulated on tumor and proliferating endothelium, partly by hypoxia and also in response to VEGF-A itself. VEGF R1 and VEGF R2 can interact with multiple downstream signaling pathways via proteins such as PLC, Ras, Sic, Nck, PKC and PI3-kinase. VEGF R1 is of higher affinity than VEGF R2 and mediates motility and vascular permeability. VEGF R2 is necessary for proliferation.
The therapeutic implications of angiogenic growth factors were first described by more than 30 years ago. (Folkman, N. Engl. J. Med., 85:1182-1186 (1971)). Recent work has established the feasibility of using recombinant angiogenic growth factors, such as vascular endothelial growth factor (VEGF-1) to expedite and/or augment collateral artery development in animal models of myocardial and hindlimb ischemia (Takeshita, et al., Circulation, 90:228-234 (1994) and Takeshita, et al., J Clin Invest, 93:662-70 (1-994)). However, alternative methods for promoting angiogenesis are desirable for a number of reasons. For example, it is believed that native endothelial cell number and/or viability decreases over time. Thus, in certain patient populations, e.g., the elderly, cells capable of responding to angiogenic proteins may be limited. These problems can be reduced by administering isolated endothelial cells to patients undergoing treatment for ischemic disease. However, prior to the present invention, such methods were prohibitively expensive as they required expensive isolation and maintenance of cells.
Angiogenesis or angiogenic capability refers to the capacity of an endothelial cell to generate new capillary blood vessels. Highly angiogenic endothelial cells have a wide spectrum of uses including, use in the prevention or treatment of trauma, graft rejection, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, ischemia related to infection, limb ischemia, ischemic cardiomyopathy, cerebrovascular ischemia, and myocardial ischemia. Impacted tissue can be associated with nearly any physiological system including the circulatory system or the central nervous system, e.g., a limb, graft (e.g., muscle or nerve graft), or organ (e.g., heart, brain, kidney and lung). Highly angiogenic endothelial cells may also be utilized in angiogenesis assay kits and in the study of endothelial cells, particularly the functions and permeability of the endothelial cell barrier. In order to determine angiogenic capability and the ability of endothelial cells to form tubes it has been necessary to carry out angiogenesis assays for each lot of endothelial cells. These assays are time consuming and costly. Therefore, there is a need for a cheaper, quicker way to screen for and mark angiogenesis capability among endothelial cells. This need is met by the present invention which provides an effective and cost-efficient means of identifying highly angiogenic endothelial cells.